ARTICLE IN PRESS
Journal of Insect Physiology 53 (2007) 708–714
www.elsevier.com/locate/jinsphys
Mating behavior of the hematophagous bug Triatoma infestans:
Role of Brindley’s and metasternal glands
J.G. Crespo, G. Manrique
Laboratorio de Fisiologı´a de Insectos, Departamento de Biodiversidad y Biologı´a Experimental, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Ciudad Universitaria, Pabellon II, Buenos Aires C1428EHA, Argentina
Received 11 August 2006; received in revised form 23 March 2007; accepted 26 March 2007
Abstract
We investigated if Brindley’s and metasternal glands are involved in the sexual behavior of Triatoma infestans. In laboratory assays, we
analyzed the effect of selective occlusion of Brindley’s and metasternal glands of the female (separately and together) on the behavior of
males. Control assays without occlusion of glands were also performed. We quantitatively tested if such glands affect mating occurrence,
the copulatory attempts of males, and the aggregation of males around a mating couple. The number of mating attempts by males did
not differ between treatments, demonstrating that likelihood of males mating did not depend on which gland is occluded in the female. In
the absence of any occlusion, T. infestans mated and males aggregated. The proportion of copulations and aggregation behavior of males
did not differ between treatments when female’s Brindley’s glands were occluded. However, when metasternal glands were occluded, the
proportion of mating couples decreased and males did not aggregate. We demonstrated that the metasternal glands of the female are
involved in the sexual behavior of T. infestans, while Brindley’s glands seem to have no effect on mating behavior. Copulation and
aggregation behavior of males likely result from the eventual release of volatiles from the female’s metasternal glands.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Triatoma infestans; Metasternal glands; Brindley’s glands; Sexual behavior
1. Introduction
The mating behavior of several Triatominae (Heteroptera: Reduviidae) species has been described as a
conservative pattern consisting of a sequence of behavioral
steps performed mainly by the male (Lima et al., 1986;
Rojas et al., 1990; Rojas and Cruz-López, 1992). In
addition, female receptivity has been demonstrated to
affect mating success in Triatoma infestans Klug 1834 and
Panstrongylus megistus Burmeister 1835 (Manrique and
Lazzari, 1994; Pires et al., 2004). For instance, nonreceptive females can reject male copulatory attempts by
stridulation (Roces and Manrique, 1996; Manrique and
Schilman, 2000). The possible existence of chemical cues
affecting mating behavior of triatomines has been studied.
Behavioral evidence suggests that a volatile signal is
Corresponding author. Tel.: +54 11 4576 3300x332;
fax: +54 11 4576 3384.
E-mail address: [email protected] (G. Manrique).
0022-1910/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinsphys.2007.03.014
released during mating and promotes the assembling of
males towards the couple (Baldwin et al., 1971 in Rhodnius
prolixus Stal 1859; Manrique and Lazzari, 1995 in
T. infestans). However, the source (i.e., sex, gland) and
chemical identity of this signal remain elusive.
Adult triatomines possess two pairs of main exocrine
glands, Brindley’s and metasternal glands (Schofield and
Upton, 1978). Brindley’s glands are dorsally located,
extending into the lateral portion of the second abdominal
segment and opening to the exterior through an orifice
situated in the metathoracic episternum (Kälin and Barrett,
1975; Staddon, 1983). It had been shown that in
T. infestans the secretion from Brindley’s glands, or the
blend emitted after mechanical disturbance of bugs,
consists of isobutyric acid (Schofield, 1979), short chain
fatty acids (plus isobutyric acid) (Hack et al., 1980; Juárez
and Brenner, 1981) or a mixture of isobutyric acid and
other minor compounds, including diverse alcohols and
esters (Cruz-López et al., 1995; Guerenstein and Guerin,
2004). In this species, it was recently shown that Brindley’s
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J.G. Crespo, G. Manrique / Journal of Insect Physiology 53 (2007) 708–714
glands are also the source of a more complex mixture
containing isobutyric acid as the main component, other
short chain fatty acids, alcohols, esters, a ketone and two
aromatic compounds (Manrique et al., 2006). Moreover,
several authors suggest that this secretion may serve some
defensive function because of its corrosive nature and
alarm function, based on behavioral evidence (Schofield,
1979; Ward, 1981; Manrique et al., 2006).
The metasternal glands are ventrally located at the
anterior margin of each metacoxal cavity and are lateral to
the apophysis. The opening of each gland is also lateral to
the apophysis and to the apophyseal pit (Weirauch, 2006).
Recently, secretions of the metasternal glands of
T. infestans were found to contain 3-pentanone together
with alcohols and other compounds (Manrique et al.,
2006). Thus far, the function of these glands has not been
extensively studied. The occasional emission of 3-pentanone during copulation of T. infestans was reported by
Manrique et al. (2006), who suggest that metasternal
glands play a role in sexual communication. Fontán et al.
(2002) reported the release of isobutyric acid in quantities around 250 ng by one pair of T. infestans during
copulation. These authors detected this compound together with other fatty acids, alcohols and traces of
aldehydes. Moreover, they reported that 3-methylbutan-1-ol and several aldehydes were electrophysiologically and behaviorally active, which suggests that the
compounds may be related with the postulated copulation
pheromone.
The role of the main exocrine glands in the sexual
context of triatomines is unresolved. In this study, we
tested if Brindley’s and metasternal glands are involved in
different aspects of the sexual behavior of T. infestans
through the selective occlusion of both glands of the
female, separately and together, and analysis of the
subsequent behavior of males. We examined if such glands
affect the occurrence of mating, the number of copulatory
attempts performed by males, and the assembling of males
in presence of a mating couple.
709
2.2. Occlusion of glands
Scales of paraffin (Paraplast, Sigma) were melted and
molded over the external openings of the corresponding
pair of glands with a microcautery (WAX-PEN/HOT PEN
#2, Electron Microscopy Sciences). This technique was
shown to be efficient in preliminary assays for at least 30 h.
For this reason, all the experiments were conducted 24 h
after the paraffin treatment. It was suggested that when
adults of T. infestans are mechanically disturbed, metasternal and Brindley’s glands discharge their contents together
(Manrique et al., 2006). This disturbance produces a
repugnant odor easily detected by humans, which was
used as a qualitative indicator for the correct occlusion of
both types of females’ glands. As the wing insertions are
very close to external openings of the Bindley’s glands,
wings were carefully removed before the occlusion.
2.3. Experimental design
In each assay, four males were released in the center of a
circular glass arena (6 cm height 26 cm diameter) with a
piece of filter paper as substrate, which was replaced
between assays to avoid chemical contamination (e.g. feces)
(Fig. 1). After 3 min, a female marked with white acrylic
paint was released with the males. To avoid mechanical
disturbance that could trigger any discharge of the glands
when released into the arena, each individual was allowed
to climb onto a piece of filter paper, which was then placed
into a delivery flask. Flasks were then inverted over the
arena and carefully lifted after 3 min. Each assay lasted till
the first copula or for at least 15 min. This time was selected
taking into account that the copula of T. infestans lasts ca.
Release bowl
Video recorder
Lamp
2. Materials and methods
2.1. Insects
Fifth instar larvae of T. infestans were sorted by sex
following the methods of Espı́nola (1966). Larvae were fed
and kept in acrylic containers with a piece of filter paper as
substrate, until their imaginal ecdysis. Insects were
provided by the Servicio Nacional de Chagas (Santa Marı́a
de Punilla, Córdoba, Argentina) reared from the eggs at
2872 1C; 30–60% RH with a photoperiod of 12:12 h (L:D)
and fed in vivo on live hens. Throughout this study, we used
virgin adults between 1 and 2 months old that were prone
to mate (Manrique and Lazzari, 1994) and were starved for
3–5 days prior to the experiment. The time elapsed between
the first and last imaginal molt of the insects used did not
exceed 20 days.
Insects
Experimental arena
Fig. 1. Experimental device. Four males and one marked female were
released on the arena. The behavior of non-mating males was studied
before, during and after the copula. In order to prevent any disturbance,
we used an inverted flask raised by a nylon thread from the outside of the
device to release the insects.
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J.G. Crespo, G. Manrique / Journal of Insect Physiology 53 (2007) 708–714
10 min (Schofield, 1979) and that in preliminary assays the
insects mated during the first 5 min after they were released.
The insects’ behavior was registered by means of a video
camera (Videoman, LEE-CSA21) located 30 cm above the
arena and the video film stored for later analysis.
The experimental series were defined as a function of the
glands of the females and whether the glands were occluded
or not. The control treatment consisted of three series—C1:
intact females (N ¼ 11); C2: females with their two pairs of
wings cut off (N ¼ 12); and C3: sham females, i.e., with
paraffin placed around the secretion openings of both pairs
of glands but without occlusion (N ¼ 11). There were three
occlusion treatments—B: females with their two pairs of
wings cut off and with the secretion openings of Brindley’s
glands occluded (N ¼ 12); M: females with their two pairs
of wings cut off and with the secretion openings of
metasternal glands occluded (N ¼ 26); and M+B: females
with their two pairs of wings cut off and with the secretion
openings of Brindley’s and metasternal glands occluded
(N ¼ 24). All assays were performed at the beginning of the
scotophase, which is close to the main activity burst found
in this nocturnal species (Lazzari, 1992). Room temperature was maintained at 2574 1C, 4378% RH and a
homogeneous illumination intensity of 3573 lx was set.
2.4. Data analysis
In all series we quantified: (a) ‘‘mating frequency’’ as the
proportion of assays in which copula occurred. A copulation, or successful mating attempt, was registered if male’s
and female’s genitalia were in contact; (b) ‘‘mating
attempts frequency’’ as the number of times a male tried
to copulate with a female per assay. A mating attempt was
counted every time a male jumped or climbed onto a
female, thus mating attempts frequency includes both
successful (copula) and unsuccessful attempts; (c) ‘‘males’
aggregation index’’ as the mean distance between nonmating males and the mating couple. The ‘‘males’
aggregation index’’ was computed before, during and after
the copula, measuring the distance of each male in the
arena to the female (center of pronotum as reference) at
1-min intervals. A program (Análisis de video 1.0) designed
ad hoc in our laboratory allowed us to digitalize the x–y
coordinates of each bug and to calculate the distances
between individuals.
As no differences were found between the ‘‘mating
frequency’’, ‘‘mating attempt frequency’’ and ‘‘male
aggregation index’’ of the first three series (C1, C2 and
C3), they were grouped and named as ‘‘control series’’ (C).
‘‘Mating frequencies’’ were analyzed and compared by
means of a G-test for goodness of fit and nonsignificant
subsets to compare between treatments (BIOMstat 3.2).
‘‘Mating attempts’’ of males were analyzed by means of
one-way analysis of variance (ANOVA) after verifying the
assumptions, i.e., normal distribution of residuals and
homogeneity of variance (Bartlett test). A regression
analysis was used to describe the relationship between the
duration of the copula and the ‘‘male aggregation index.’’
For each experiment, we calculated the slope of each curve
and used a one-way ANOVA analysis to test for the effect
of treatment, after verifying the assumptions, i.e., normal
distribution of residuals and homogeneity of variance
(Bartlett test). According to this analysis, males are
considered to be aggregating near the mating pair if there
is a decrease in the relative distance of males around the
mating couple for as long as the copula lasts (i.e., slope is
negative). The LSD test was used for comparisons a
posteriori between treatments (Zar, 1984).
3. Results
3.1. Mating frequency
Fig. 2 depicts the percentage of assays in which
copulation occurred during the different experimental
series performed. Statistical analysis revealed highly
significant differences among treatments, showing that
the different treatments modified the mating frequency of
these bugs (G ¼ 28.2; df ¼ 3; po5E6). The a posteriori
comparison revealed no significant differences between the
control (C) and Brindley’s occluded glands series (B)
(p40.05). In both cases, about 80% of the couples
copulated. The groups of insects in which the female had
the metasternal glands occluded (M) and both glands
occluded (M+B) presented a similar frequency of copulation (p40.05), around 35%. However, a significant
decrease in mating frequency was observed when insects
(34)
100
Mating proportion (%)
710
a
(12)
a
80
60
(26)
b
40
(24)
b
20
0
C
M
B
Treatments
B+M
Fig. 2. Percentage of mating pairs (number of assays in which copula
occurred/ total number of assays) according to the treatment applied on
the female—C (control): control females (no occlusion at all); B: females
with the secretion openings of Brindley’s glands occluded; M: females with
the secretion openings of the metasternal glands occluded; B+M: females
with the secretion openings of both kinds of exocrine glands occluded.
High significant differences were found between all the experimental
groups (G ¼ 28.2; df ¼ 3; po5E6). Different letters represent significant
differences between series (a posteriori comparisons). Sample sizes are
indicated between brackets.
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J.G. Crespo, G. Manrique / Journal of Insect Physiology 53 (2007) 708–714
with metasternal glands occluded (either M or M+B
series) were compared to insects without these glands
occluded (i.e., C and B series) (po0.05, in all cases).
711
1.770.1 mating attempts per male) regardless of which
glands are occluded on the female.
3.3. Male aggregation index
3.2. Mating attempt frequency
We studied the effect of the secretions of both kinds of
exocrine glands on the assembling behavior of males
around mating pairs, previously described for this species
(Manrique and Lazzari, 1995). Analysis of selective
occlusion of each pair of glands separately and together
on females and the subsequent distance of males from the
copulating pair revealed significant differences among the
spatial distribution of males in the different treatments,
thus revealing that the differential occlusion of female’s
glands modified the behavior of surrounding males
(ANOVA, F(3, 54) ¼ 8.79; po0.0001). Fig. 3 shows the
mean distance of males towards the mating pair for all
treatments, from the beginning (t ¼ 0) till the end of
mating (separation of genitalia). Males from the control
series (C) (Fig. 3A), as well as those in the series where the
Brindley’s glands of the female were occluded (B) (Fig. 3B),
presented a decrease in their mean distance towards the
mating pair as a function of time. In addition, we found no
significant differences between these two series (LSD test,
p40.05). When we quantified the aggregation around a
copulating pair where the female had either its metasternal
(M) (Fig. 3C) or both exocrine glands occluded (M+B)
(Fig. 3D), males did not aggregate. In addition, no
significant differences between these two series were found
Given the low mating frequency of couples when the
female’s metasternal glands were occluded (series M and
M+B), we tested if this was caused by males’ reluctancy to
mate. Table 1 shows the mean of successful and
unsuccessful mating attempts of males per assay for each
experimental series. No significant differences were found
among treatments (ANOVA, F(3,92) ¼ 0.93; p40.05).
Males attempt to copulate at similar rates (mean7S.E.:
Table 1
Mean7S.E. of the successful and unsuccessful mating attempts of males
per assay registered in each experimental series
Treatments
Mean7S.E. of male mating
attempts per assay
Replicates
C
B
M
B+M
1.770.3
1.970.4
1.970.2
1.570.2
34
12
26
24
No significant differences were found between treatments (ANOVA,
F(3, 92) ¼ 0.93; p40.05). C: control females; B: females with the secretion
openings of Brindley’s glands occluded; M: females with the secretion
openings of the metasternal glands occluded; and B+M: females with the
secretion openings of both kinds of exocrine glands occluded
Control
16
14
(30)
12
12
10
10
8
8
6
6
Mean distance (cm)
Mean distance (cm)
14
4
2
0
16
Brindleys glands occluded
(10)
14
Metasternal glands occluded
(10)
16
12
4
2
0
both glands occluded
(8)
16
14
12
10
10
8
8
6
6
4
4
2
2
0
0
0
1
2
3
4
Time (min)
5
6
7
0
1
2
3
4
5
6
7
Time (min)
Fig. 3. Mean distance7S.E. between non-mating males and the mating pair as a function of time: (A) control females; (B) females with the secretion
openings of Brindley’s glands occluded; (C) females with the secretion openings of the metasternal glands occluded; (D) females with the secretion
openings of both exocrine glands occluded. The dotted line indicates the beginning of the copula (t ¼ 0). Series C and B did not differ significantly,
evincing a tendency of males to aggregate around the mating pair (LSD test, p40.05) (parts (A) and (B)). Neither did Series M and B+M (LSD test,
p40.05) (parts (C) and (D)). However, when C and B series were compared with M and B+M, significant differences were found (LSD test, po0.05).
Sample sizes are indicated between brackets.
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J.G. Crespo, G. Manrique / Journal of Insect Physiology 53 (2007) 708–714
(LSD test, p40.05). However, when we compared the first
two series (C and B) (Fig. 3A and B) with the lasts two
series (M and M+B) (Fig. 3C and D), we found
statistically significant differences between them, revealing
that occluding the metasternal glands prevents the assembling behavior of males (LSD test, po0.05 in all cases).
4. Discussion
In the present work, we demonstrated that the metasternal glands of the T. infestans female are involved in
the sexual behavior of this species, promoting mating and
the aggregation of males towards a mating couple. On the
other hand, under our experimental conditions, Brindley’s
glands did not affect mating behavior.
Previous studies suggested a relationship between several
Brindley’s glands products and the sexual behavior of
triatomines (Fontán et al., 2002; Rojas et al., 2002;
Guerenstein and Guerin, 2004). In addition, Fontán et al.
(2002) reported that traces of aldehydes were found over
mating pairs of T. infestans and demonstrated that these
substances were electrophysiologically and behaviorally
active. Nevertheless, to date no aldehyde-producing glands
are known in triatomines and therefore no sources other
than hosts (Guerenstein and Guerin, 2001) can be
suggested for the origin of those compounds. Moreover,
the behavioral responses recorded testing aldehydes are not
sex-specific behaviors and do not exclude attraction to
other kind of stimuli, as host odors.
We based our research in experiments of selective
occlusion of the glands, in order to analyze the behavioral
responses evoked by each gland. The results presented here
show that the proportion of copulations and the aggregation behavior of males did not differ from control insects
(C) when Brindley’s glands of females were occluded (B).
This fact suggests that the volatile compounds of the
female’s Brindley’s glands do not have a role either in
mating occurrence or in the promotion of the aggregation
behavior of T. infestans males. However, to fully test this
hypothesis, it would be valuable to analyze the specific
compounds and combinations of them in the sexual
context. On the other hand, we clearly demonstrated here
that the female’s metasternal glands have an important role
in the sexual activity of these insects, since, when occluded
(M and M+B), the proportion of mating couples and the
aggregation behavior of males was affected. On the
contrary, in the experiments where the female had its
metasternal glands free of occlusion, bugs mated and
aggregated in a similar fashion as control insects (C and B).
Therefore, copulation and the aggregation behavior of
males can be ascribed to the release of volatile compounds
from the female’s metasternal glands. These results are
supported by the recent detection of 3-pentanone, the main
constituent of metasternal glands of T. infestans, released
from copulating pairs (Manrique et al., 2006).
We have yet to determine in which stage of the male precopula behavior the presumed chemical signal would be
acting. However, it can be speculated that this signal acts
once the male physically contacts the female. This
supposition is based on the observation that in presence
of a female with its metasternal glands occluded, males
behave normally, i.e., perform the behavioral phases in a
similar way as control males, at least until the male
contacts the female. After such contact, the occurrence of
copulation is affected. In addition, the presence of other
possible chemical signals like cuticular hydrocarbons, as
occurs in other insect species (e.g., in the coleopteran
Anoplophora malasiaca Thompson; Fukaya, 2003), may
affect the attraction and/or sexual recognition; and even
the utilization of other cues of different modality (e.g.,
visual, mechanical). Other glandular areas described by
Barth (1980) and Weirauch (2003, 2004) should also be
considered. Because fewer copulations are observed when
the metasternal glands of the females were occluded
compared to controls, it is relevant to ask if the male’s
mating attempts are responsible of this decrease. If males
cause a lower proportion of copulation, the number of
successful and unsuccessful mating attempts in the treatment series should differ from control insects. However, we
found that the decrease in the mating proportion registered
in the assays with metasternal glands occluded was not due
to reluctance of the males to mate, since this treatment had
no effect on the motivation of males to mate. A similar
result was obtained when the female’s Brindley’s glands
were occluded, demonstrating again that males are prone
to mate under this conditions. Moreover, it is corroborated
that the decrease in mating occurrence was not due to the
experimental manipulation.
The mating and aggregation behavior of males is not
consistent across all triatomines. P. megistus females are
receptive to copulation once or very few times during their
adult life, perhaps because the female remains united with
the male through their genitalia for a long time thus
avoiding mating with other males (Pires et al., 2004). These
authors also showed the apparent absence of signals
promoting the aggregation of males in presence of a
mating pair; in contrast to what occurs in T. infestans and
R. prolixus (Baldwin et al., 1971; Manrique and Lazzari,
1995). In T. infestans, the couple remains united for a
shorter time, which may allow for subsequent copulations.
Some preliminary experiments show that aggregated males
copulated successively with the same female (Manrique and
Lazzari, 1995). Thus, the sexual signal emitted by the
female’s metasternal glands, which promotes the aggregation of males, could be related to the existence of a
polyandrous mating system (Thornhill and Alcock, 1983).
Because the attractiveness of mating pairs decreases when
the female has occluded metasternal glands, we suggest
that the female emits the volatile compounds involved in
promoting both copulation and aggregation behavior of
males. In T. infestans, females were not observed to
aggregate around a mating couple, suggesting that female
behavior is not influenced by the presence of a copulating
pair (Manrique and Lazzari, 1995). However, the potential
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emission of volatiles from males influencing sexual
behavior cannot be discarded, as this apparently occurs
in R. prolixus (Barcelos Ponte, G. personal communication).
Interestingly, it has been demonstrated that both kinds
of glands, metasternal and Brindley’s glands, discharge
their contents during mechanical disturbance of the insect
(Manrique et al., 2006), which suggests that metasternal
glands could also be involved in alarm and/or defensive
contexts. Glands, which were clearly adapted to have a role
in sexual behavior, have been found to retain their
defensive properties in a variety of arthropods (Blum,
1996). Thus, at this time we cannot rule out the possibility
that metasternal glands have other functions besides those
involved in mating. Future electrophysiological and
behavioral studies should analyze the specific functions of
the volatiles produced by metasternal and Brindley’s
glands to determine which glands and glandular products
are involved in sexual behavior. Because these bugs are
vectors of the protozoan flagellate Trypanosoma cruzi, the
etiological agent of Chagas disease, future experiments
could use chemical manipulation of the behavior of these
bugs to provide insights into the transmission of Chagas
disease, which affects 16–18 million people in Latin
America (Dias et al., 2002; WHO, 2002).
Acknowledgments
The authors are deeply indebted to Sebastián A. Minoli
(Institut de Recherche sur la Biologie de l’Insecte, Faculté
des Sciences et Techniques, Université Franc- ois Rabelais,
France) and Marcelo G. Lorenzo (Centro de Pesquisas
René Rachou/CPqRR, Oswaldo Cruz Foundation) for
critically reading and improving the manuscript and the
staff members of our laboratory for many fruitful
discussions. We wish to thank Erin E. Wilson (University
of San Diego at California) for her advice and correction of
the English. This investigation received financial support
from the UNDP/World Bank/WHO Special Program for
Research and Training in Tropical Diseases (TDR),
CONICET and Universidad de Buenos Aires.
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